The selective action of certain antibiotics on various aspects of protein synthesis will be studied: (1) We will use purified polysomes, containing endogenous or viral natural messenger RNA, to pursue further two initial observations: that the misreading effect of streptomycin is exerted on already engaged polysomal ribosomes, and that various aminoglycosides differ in the ratio of their misreading effect on such preparations. (2) Having found that spectinomycin and erythromycin, like streptomycin, block initiating ribosomes at some step after initiation we plan to identify the blocked step for each and possibly to extend this approach to additional antibiotics. Translation by ribosomes of natural polycistronic mRNA will be studied with purified, initiation-free polysomes enriched with specific types of messenger. Polypeptides formed from these polysomes will be isolated with specific antibodies made against them. N-terminal amino acid and the size of the peptides formed from these polysomes will be analyzed, and the results should enable us to determine whether ribosomes come off at the end of a gene or the end of a mRNA. The possible existence of "precursor peptide" and "processing enzymes" will be tested for in cells with certain amino acid analogues in the presence of protease inhibitors to decrease peptide cleavage or degradation. Ribosome release factor will be purified to homogeneity, and antibodies made against it. The physiological role of this factor in the natural release of ribosomes from mRNA and in the breakdown of polysomes in the absence of protein synthesis (e.g., amino acid starvation by certain antibiotics) will be studied with purified polysomes and purified factors. BIBLIOGRAPHIC REFERENCES: Smith, W.P., Tai, P.-C., Thompson, R.T., and Davis, B.D. Extracellular labeling demonstrates nascent polypeptides traversing the membrane of E. coli. (Abstract) Fed. Proc. 36:897 (1977). Kung, H.F., Treadwell, B.V., Sprears, C., Tai, P.-C., and Weissbach, H. DNA-directed in vitro synthesis of beta-galactosidase: requirement for a ribosomes release factor. Pro. Nat. Acad. Sci. USA, in press (1977).